Functional characterization of a piscine retroviral promoter

Walleye dermal sarcoma virus (WDSV) is a retrovirus aetiologically associated with a multifocal skin tumour of walleye. Tumours synchronously develop on 27% of fish and regress seasonally; their severity is influenced by water temperature. To functionally characterize the LTR of WDSV, the LTR was fused to the luciferase reporter gene. WDSV LTR was found to be transcriptionally active in both fish and mammalian cells. WDSV LTR deletion mutants were constructed to identify specific regions that were functionally important in modulating viral gene expression and in temperature responsiveness. The 5' end 60 bp, which contain a putative ecdysone-response element also present in another fish retrovirus, positively modulated transcription from the WDSV LTR at 25 degrees C, but not at lower temperatures. A 13 bp region (nt -288 to -275) comprising a putative activator protein-1 element was necessary for maintaining WDSV LTR activity at all temperatures. In marked contrast to the short direct repeats found in mammalian retroviral LTRs, five 5 bp direct repeats (nt -336 and -272) were found to negatively regulate transcription from the WDSV LTR. A region spanning nt -440 to -218 stimulated the activity of a heterologous mammalian promoter in an orientation-dependent manner, modestly in fish cells (1.3- to 2-fold), but markedly (3.7- to 5.1-fold) in mammalian cells. Our results strongly suggest that the putative promoter elements present in the WDSV LTR function differentially in a temperature-specific manner and that complex interactions between these elements modulate WDSV LTR activity in response to temperature changes.